Stereocilia, the key cellular organelles responsible for auditory and vestibular sensory function, are organized into bundles of precisely specified rows of increasing heights forming characteristic staircase patterns. Although stereocilia are exquisitely sensitive to mechanical vibration, orderly structured, and easily damaged by over stimulation, they are maintained in proper working order for an entire lifetime. Each stereocilium is supported by a rigid paracrystalline array of several hundred parallel, uniformly polarized and regularly cross-linked actin filaments. We have previously shown that the seemingly static actin paracrystal at the core of sensory stereocilia of hair cells undergoes continuous renewal by reproducing itself at the stereocilia tips, treadmilling rearwards, and dismantling itself at the base (Schneider et al. Nature, 418: 837, 2002). We have now used the same approach of transfecting hair cells with actin-green fluorescent protein (GFP), espin-GFP, and myosin XVa-GFP to characterize the turnover process. Actin and espin are incorporated at the paracrystal tip and flow rearwards at the same rate. The flux rates (~0.002?0.04 actin subunits per second) were proportional to the stereocilia length so that the entire staircase stereocilia bundle was turned over synchronously. Actin polymerization inhibition by cytochalasin D caused stereocilia to shorten at rates matching paracrystal turnover. Myosins Ic and VIIa were localized alongside the actin paracrystal where they could drive retrograde actin flow. Myosin XVa was observed at the stereocilia tips, coinciding with a structure called the tip density, at levels proportional to stereocilia lengths. Electron microscopy analysis of the abnormally short stereocilia in the shaker 2 (myosin XVa mutant) mice did not show the characteristic tip density structure. We also show that stereocilia length is modulated by the expression levels of espin and myosin XVa as well as by local physical parameters, such as tension on the stereocilia links and on the encapsulating membrane. We argue that regulation of actin polymerization and treadmilling dynamically shapes the functional architecture of stereocilia and plays a central role in recovery from over-stimulation.